khavarilabfandomcom-20200213-history
Laser capture microdissection
From VLP 'Cryosectioning' *Clean cryostat with EtOH to make RNase-free *Use PEN-foil membrane slides (Leica #11505158, order from JT Technologies) *Cut at 7-10µm thickness (7µm best) *Can place 7-10 sections per slide *Need ~8 slides per sample (depending on what you’re isolating) *Keep slides on dry ice or in cryostat AT ALL TIMES *Store slides at -80ºC—keep as dry as possible *'Slides should be used within 4 days of sectioning' 'Staining:' *Use LCM Staining Kit (Ambion #AM1935) *Follow protocol that comes with kit with minor modifications (work quickly to rehydrate/dehydrate tissue) *Setup: Fill chambers with ~30ml EtOH and place in 50ml Falcon tube rack; make paper towel blotter; get Cresyl violet out of 4ºC walk-in *Briefly: *Take slides out of -80ºC and place on dry ice *Quickly place in 95% EtOH 30-40s while moving slides up and down with forceps and remove excess EtOH by tapping on kimwipe *Transfer slides to 75%EtOH chamber for 30-40s while moving slides up and down and remove excess EtOH by tapping on kimwipe *Transfer slides to 50%EtOH chamber for 25-30s while moving slides up and down and remove excess EtOH by tapping on kimwipe *Mark area to be stained with barrier pen making sure not to touch foil area *Cover area with 300-800µl cresyl violet stain for up to 1.5min *Dump stain on paper towel blot and tap on kimwipe *Place stained slide in 50%EtOH chamber for 30-40s and move up and down several times, remove excess on kimwipe *Transfer slides to 75%EtOH chamber for 30-40s and move up and down several times, remove excess on kimwipe *Transfer slides to 95%EtOH chamber for 30-40s and move up and down several times, remove excess on kimwipe *Transfer slide to 100%EtOH for 30-40s, remove excess EtOH on kimwipe *Transfer slides to 2nd 100%EtOH chamber for up to 2 min, remove excess EtOH on kimwipe *Transfer slide to xylene moving up and down to remove EtOH *Transfer slide to 2nd xylene chamber and incubate for 5 min *Remove slides from xylene and air-dry in hood against box for 10 min 'LCM': *Turn on microscope in ordered labeled (controller 1st, laser 2nd) *Turn on computer and open “Leica Microdissection 4.4” software *Make sure tube and slide holder are not in *Clean tube and slide holder with RNase-zap *Place slide upside-down in slide holder *Click “unload” iconàplace slideholder in and click into place *Fill tube holder with PCR tubes and push into place under slide (snaps into place) *Click “continue” on open window *Use the controller to change magnification, focus, move platform, adjust lighting (Only use the controller to modify these settings) *Calibrate the laser in the objective used for cutting: Click on the “Laser” drop-down menuà “calibrate” (make sure laser is not near tissue)àline up arrows for calibration *Bring PCR tube used for collection by selecting icon at bottom of screen—cap should go from red to green and color of selection line will match cap letter color *Click on “line” iconàselect “closed lines” and “combined mode”à “start cut” *If section cut does not fall into cap, use the “move and cut” function to manually direct the laser—'may need to refocus to properly cut' *To check if cut section fell into cap, click on “collector” icon to inspect cap—should see small pieces of tissue (Do not change objective in this mode) *To change intensity, speed or thickness of laser, click on “Laser” drop-down menu and “control” *Click on “unload” icon to change slides or PCR tubes *'CAREFULLY' remove PCR tube from tube holder (can add lysis buffer to cap prior to removal) *Place tissue on dry ice and store at -80ºC or immediately extract RNA *''For human epidermis, need to collect 500K-1000K µm2 for ~50-100ng RNA'' 'RNA Isolation:' *Use RNAqueous-Micro kit (Ambion #AM1931) *Follow kit protocol, Section B: RNA isolation protocol from LCM sampes *Modifications: *Step 4: add 1.25 volumes 100% EtOH *Step 9: elute with 2x8µl elution solution *Store at -80ºC 'RNA Amplification:' Use 10µl RNA per reaction (should get ~10-12µl from RNA isolation step—generally use all RNA for amplification—may need to do 2 rounds) Include DNase treatment after in vitro transcription if downstream application is qRT-PCR